Viral vectors and their use in therapeutic methods

ABSTRACT

The invention provides viral vectors (e.g., herpes viral vectors) and methods of using these vectors to treat disease.

CROSS REFERENCE TO RELATED APPLICATIONS

This application is a divisional of, and claims priority from, U.S.patent application Ser. No. 12/721,599, filed Mar. 11, 2010 (U.S. Pat.No. 8,470,577), which is a continuation of U.S. patent application Ser.No. 10/107,036, filed Mar. 27, 2002 (U.S. Pat. No. 7,749,745), whichclaims benefit of U.S. Provisional Patent Application No. 60/279,069,filed Mar. 27, 2001, each of which is incorporated herein by reference.

FIELD OF THE INVENTION

This invention relates to viruses and their use in therapeutic methods.

BACKGROUND OF THE INVENTION

The use of replication-competent viral vectors, such as herpes simplexvirus type 1 (HSV-1) vectors, is an attractive strategy for tumortherapy, because such viruses can replicate and spread in situ,exhibiting oncolytic activity through direct cytopathic effect (Kim, J.Clin. Invest. 105:837-839, 2000). A number of oncolytic HSV-1 vectorshave been developed that have mutations in genes associated withneurovirulence and/or viral DNA synthesis, in order to restrictreplication of these vectors to transformed cells and not cause disease(Martuza, J. Clin. Invest. 105:841-846, 2000).

In designing viral vectors for clinical use, it is essential that amplesafeguards be employed. G207 is an oncolytic HSV-1 vector derived fromwild-type HSV-1 strain F (Mineta et al., Nat. Med. 1:938-943, 1995). Ithas deletions in both copies of the major determinant of HSVneurovirulence, the 734.5 gene, and an inactivating insertion of the E.coli lacZ gene in UL39, which encodes the infected-cell protein 6 (ICP6)(Mineta et al., Nat. Med. 1:938-943, 1995). ICP6 is the large subunit ofribonucleotide reductase, a key enzyme for nucleotide metabolism andviral DNA synthesis in non-dividing cells but not dividing cells(Goldstein et al., J. Virol. 62:196-205, 1988). In addition to being themajor determinant of HSV neurovirulence (Chou et al., Science250:1262-1266, 1990), ICP34.5 also functions by blocking host cellinduced shutoff of protein synthesis in response to viral infection(Chou et al., Proc. Natl. Acad. Sci. U.S.A. 89:3266-3270, 1992). This islikely responsible for the less efficient growth of γ34.5⁻ mutantscompared to wild-type HSV, which has been observed in many tumor celltypes (McKie et al., Br. J. Cancer 74:745-752, 1996; Andreansky et al.,Cancer Res. 57:1502-1509, 1997; Chambers et al., Proc. Natl. Acad. Sci.U.S.A. 92:1411-1415, 1995). This double mutation confers importantadvantages: minimal chance of reverting to wild type, preferentialreplication in tumor cells, attenuated neurovirulence, andganciclovir/acyclovir hypersensitivity. G207 effectively kills multipletypes of tumor cells in culture and in mice harboring tumorssubcutaneously or intracranially (Mineta et al., Nat. Med. 1:938-943,1995; Yazaki et al., Cancer Res. 55:4752-4756, 1995; Toda et al., Hum.Gene Ther. 9:2177-2185, 1998; Todo et al., Hum. Gene Ther. 10:2741-2755,1999; Chahlavi et al., Neoplasia 1:162-169, 1999; Kooby et al., FASEB J.13:1325-1334, 1999; Lee et al., J. Gastrointest. Surg. 3:127-133, 1999).In several syngeneic tumor models in immunocompetent mice, oncolysiscaused by intraneoplastic inoculation of G207 elicited a systemic immuneresponse and tumor-specific cytotoxic T lymphocytes (Todo et al., Hum.Gene Ther. 10:2741-2755, 1999; Toda et al., Hum. Gene Ther. 10:385-393,1999; Todo et al., Hum. Gene Ther. 10:2869-2878, 1999).

G207 has minimal toxicity when injected into the brains ofHSV-1-susceptible mice or nonhuman primates (Hunter et al., J. Virol.73:6319-6326, 1999; Sundaresan et al., J. Virol. 74:3832-3841, 2000;Todo et al., Mol. Ther. 2:588-595, 2000). Recently, G207 has beenexamined in patients with recurrent malignant glioma (Markert et al.,Gene Ther. 7:867-874, 2000), and the results from this phase I clinicaltrial indicate that intracerebral inoculation of G207 is safe at dosesof up to 3×10⁹ plaque forming units (pfu), the highest dose tested.While the use of oncolytic viruses is a promising approach for cancertherapy, the therapeutic benefits will likely depend on the dose androute of administration, the extent of intratumoral viral replication,and the host immune response.

HSV-1 infection causes down-regulation of major histocompatibilitycomplex (MHC) class I expression on the surface of infected host cells(Jennings et al., J. Virol. 56:757-766, 1985; Hill et al., J. Immunol.152:2736-2741, 1994). The binding of ICP47 to the transporter associatedwith antigen presentation (TAP) blocks antigenic peptide transport inthe endoplasmic reticulum and loading of MHC class I molecules (York etal., Cell 77:525-535, 1994; Hill et al., Nature 375:411-415, 1995; Frühet al., Nature 375:415-418, 1995). The binding of ICP47 isspecies-specific for TAPs from large mammals (Jugovic et al., J. Virol.72:5076-5084, 1998), with the affinity for murine TAP about 100-foldless than for human (Ahn et al., EMBO J. 15:3247-3255, 1996).

SUMMARY OF THE INVENTION

The invention provides herpes simplex viruses (e.g., HSV-1 viruses) thatinclude mutations within the BstEII-EcoNI fragment of the BamHI xfragment of the viruses. These viruses can also include, for example, aninactivating mutation in the γ34.5 neurovirulence locus of the viruses,and/or an inactivating mutation in the ICP6 locus of the viruses.

Also included in the invention are herpes simplex viruses (e.g., anHSV-1 virus) that include an inactivating mutation in the ICP47 locus ofthe viruses, in the absence of an inactivating mutation in the γ34.5neurovirulence locuses of the virus. Optionally, these viruses also caninclude an inactivating mutation in the ICP6 locus of the viruses.

The invention also provides methods of inducing a systemic immuneresponse to cancer in a patient, which involve administering to thepatient a herpes virus that includes an inactivating mutation in theICP47 locus of the herpes virus. The herpes virus can be administered,for example, to a tumor of the patient. In addition, the patient canhave or be at risk of developing metastatic cancer, and the treatmentcan be carried out to treat or prevent such cancer. The inactivatingmutation in the ICP47 locus of the herpes virus can be, for example, inthe BstEII-EcoNI fragment of the BamHI x fragment of the virus.Optionally, the virus can include an inactivating mutation in the γ34.5neurovirulence locus of the herpes virus, and/or an inactivatingmutation in the ICP6 locus of the herpes virus.

The invention also provides herpes viruses that include a first mutationthat inactivates the γ34.5 neurovirulence locus of the viruses and asecond mutation that results in early expression of US11, in the absenceof an ICP47-inactivating mutation in the BamHI x fragment of theviruses. Early expression of US11 can be achieved, for example, byinserting a promoter upstream from the US11 gene, or by inserting a US11gene under the control of an early-expressing promoter into the genomeof the virus. The viruses can also include a mutation that results indownregulation of ICP47 expression, in the absence of a mutation in theBamHI x fragment of the virus. The downregulation of ICP47 can be dueto, for example, a deletion in, or inactivation of, the ICP47 promoter,or the fusion of ICP47 with a peptide that prevents functionalexpression of ICP47.

The invention also includes a herpes virus that includes a firstmutation that inactivates the γ34.5 neurovirulence locus of the virusand a second mutation that results in downregulation of ICP47expression, in the absence of a mutation in the BamHI x fragment of thevirus. The downregulation of ICP47 can be due to, for example, adeletion in, or inactivation of, the ICP47 promoter, or the fusion ofICP47 with a peptide that prevents functional expression of ICP47.

The viruses described above can also include an additional mutation(e.g., a mutation in the ICP6 locus) to prevent reversion to wild type.The viruses can also include, optionally, sequences encoding aheterologous gene product, such as a vaccine antigen or animmunomodulatory protein. The viruses described herein can be herpessimplex viruses (HSV), such as herpes simplex-1 viruses (HSV-1).

The invention further provides pharmaceutical compositions that includeany of the viruses described herein and a pharmaceutically acceptablecarrier, adjuvant, or diluent, as well as methods of treating cancer ina patient, involving administering such a pharmaceutical composition tothe patient. Also included in the invention are methods of immunizing apatient against an infectious disease, cancer, or an autoimmune disease,involving administering such a pharmaceutical composition to thepatient.

The invention provides several advantages. For example, the viruses ofthe invention replicate in, and thus destroy, dividing cells, such ascancer cells, while not affecting other cells in the body. An additionaladvantage of the viruses of the invention in which ICP47 is deleted isthat the immune response induced by such viruses is enhanced, whichresults in a better antitumor immune response. The viruses of theinvention also include multiple mutations, eliminating the possibilityof reversion to wild type. Moreover, although the viruses of theinvention may have enhanced replication, this is not accompanied byincreased toxicity. In addition, replication of herpes simplex virusescan be controlled through the action of antiviral drugs, such acyclovir,which block viral replication. These features render the viruses of theinvention to be not only effective, but safe as well.

Other features and advantages of the invention will be apparent from thefollowing detailed description, drawings, and claims.

BRIEF DESCRIPTION OF THE DRAWINGS

FIGS. 1A-1E are schematic representations of the HSV-1 genome andapproaches to making vectors included in the invention. FIG. 1A is aschematic representation of the HSV-1 genome. FIG. 1B is an expanded mapof the ICP47 locus, showing the locations of the overlapping 3′co-terminal transcripts for US10, US11, and US12 (ICP47). FIG. 1C is aschematic representation of plasmid plE12, which contains an 1818basepair BamHI-EcoRI fragment from the HSV-1 BamHI x fragment, whichencompasses the ICP47 region (Johnson et al., J. Virology68(10):6347-6362, 1994). This plasmid can be used to introducemodifications into the ICP47 locus of the viral genome, as is describedfurther below. FIG. 1D is a schematic representation of plasmid plE12 Δ,which was derived from plE12 by deleting 312 basepairs between theindicated BstEII and NruI sites. This plasmid was used to generate theγ34.5 suppressor mutants R47Δ, and G47Δ. FIG. 1E is a schematicrepresentation of the details of the 3′ terminus of the ICP47 codingregion (SEQ ID NO:1). Sequences can be inserted into the indicatedBstEII site, without disrupting sequences between the BstEII and NruIsites, for the purposes of changing the temporal regulation of the lateUS11 gene, to generate a γ34.5 suppressor function, and/or preventingfunctional expression of the ICP47 gene product.

FIGS. 2A-2C are schematic representations of the structure of G47Δ. FIG.2A is a schematic of the HSV-1 genome showing the regions modified inG47Δ. The HSV-1 genome consists of long and short unique regions (U_(L)and U_(S)), each bounded by terminal (T) and internal (I) repeat regions(R_(L) and R_(S)). The parental virus G207 was engineered from wild-typeHSV-1 strain F by deleting 1 kilobase within both copies of the γ34.5gene, and inserting the E. coli lacZ gene into the ICP6 coding region.G47Δ was derived from G207 by deleting 312 basepairs from the ICP47locus, as indicated. FIG. 2B is a map of the ICP47 locus, showinglocations of the overlapping 3′ co-terminal transcripts (US10, US11, andICP47), open reading frames (thick arrow), and ICP47 splice junctions(^). FIG. 2C is a map of plasmid pIE12Δ, which was used to generatedeletions by homologous recombination with the indicated flankingsequences. While US11 is regulated as a true late gene in wild-typeHSV-1, deletion between the indicated BstEII and EcoNI sites places US11under control of the ICP47 immediate-early promoter. Restriction siteabbreviations are: B, BamHI; Bs, BstEII; E, EcoRI; EN, EcoNI; Nr, NruI.

FIG. 3 is a graph showing virus yields of replication-competent HSV-1mutants in various cell lines. Cells were seeded on 6 well plates at5×10⁵ cells/well. Triplicate wells were infected with R3616, R47Δ, G207,G47Δ, or strain F at a MOI of 0.01. At 24 hours post-infection, cellswere scraped into the medium and progeny virus was titered on Verocells. In all cell lines tested, G47Δ showed a significantly higherreplication capability than G207. Results represent the mean oftriplicates±SD.

FIG. 4 is a series of graphs showing the cytopathic effect of G47Δ invitro. Cells were plated into 6 well plates at 2×10⁵ cells/well. After24 hours of incubation, cells were infected with G207 or G47Δ at a MOIof 0.01 or 0.1, or without virus (Control). The number of survivingcells was counted daily and expressed as a percentage of mock-infectedcontrols. G47Δ exhibited a significantly stronger cytopathic effect thanG207 in all three human tumor cell lines (U87MG and melanomas 624 and888) at a MOI of 0.01, and also in Neuro2a murine neuroblastoma cells ata MOI of 0.1. The results are the mean of triplicates±SD. * p<0.05, **p<0.01, *** p<0.001, G207 versus G47Δ, unpaired t test.

FIGS. 5A-5C are a series of graphs showing that G4F7Δ precludesdown-regulation of MHC class I expression in infected host cells. FIG.5A is a graph of flow cytometric analyses of MHC class I expression inDetroit 551 human fibroblast cells 48 hours after infection with HSV-1(MOI=3). While all HSVs with an intact α47 gene (wild-type strain F andG207) significantly down-regulated MHC class I expression, G47Δcompletely precluded the down-regulation. FIG. 5B is a graph showing atime course of MHC class I down-regulation in Detroit 551 cells infectedwith HSV-1. For each virus, the peak value of MHC class I expression at6, 24, or 48 hours post-infection, analyzed by flow cytometry, wasexpressed as a percentage of the peak value of mock-infected cells(control) at each time point. MHC class I down-regulation by G207 andR3616 occurred in a time-dependent fashion. Dissociation of MHC class Iexpression between α47-deleted mutants (G47Δ and R47Δ) and α47-intactviruses became apparent at 24-48 hours post-infection. FIG. 5C is aseries of graphs showing flow cytometric analyses of MHC class Iexpression in human melanoma cell lines 24 hours after infection withG207 and G47Δ. G47Δ caused a partial preclusion of MHC class Idown-regulation in melanomas 1102 and 938, resulting in greater MHCclass I expression than G207.

FIG. 6 is a series of graphs showing that G47Δ-infected tumor cellsstimulate T cells to a greater extent than G207-infected tumor cells.Human melanoma cells were infected with mock (no virus), G207, or G47Δat a MOI of 3, and after 3-6 hours, co-cultured with an equal number ofresponding human T cells for 18 hours. T cell stimulation was assessedby an increase in IFN-γ release into conditioned media. G47Δ-infectedmelanoma 1102 cells caused a significantly greater stimulation of TIL888cells compared with G207-infected 1102 cells (p<0.01, unpaired t test).G47Δ-infected 938 melanoma cells also stimulated TIL1413 cells, althoughthe improvement was not statistically significant compared withG207-infected 938 cells (p=0.1, unpaired t test). Neither G207 norG47Δ-infected melanoma 888 cells caused a significant stimulation ofTIL888 cells.

FIG. 7 is a set of graphs showing that G47Δ exhibits greater antitumorefficacy than G207 in vivo. Subcutaneous tumors of U87MG human glioma(Left) or Neuro2a murine neuroblastoma (Right) were generated in6-week-old female athymic mice or 6-week-old female A/J mice,respectively. Established tumors of approximately 6 mm in diameter wereinoculated with G207 or G47Δ (1×10⁶ pfu), or mock (PBS with 10%glycerol) on days 0 and 3. G47Δ treatment was significantly moreefficacious than G207 in both tumor models, resulting in smaller averagetumor volumes (p<0.05 for U87MG on day 24 and p<0.001 for Neuro2a on day15, G207 versus G47Δ, unpaired t test).

DETAILED DESCRIPTION

The invention provides viruses that can be used in therapeutic methods,such as, for example, in the treatment of cancer. These viruses areparticularly well suited for this purpose, as they replicate in, andthus destroy, dividing cells (e.g., cancer cells), but they do notreplicate substantially, and thus are avirulent, in non-dividing cells.The viruses of the invention can also be used in immunization methods,for the treatment or prevention of, for example, infectious diseases,cancer, or autoimmune diseases. An advantageous feature of many of theviruses of the invention is that, in addition to directly causing lysisof tumor cells, they induce a systemic immune response against tumors.Thus, these viruses can be used not only to treat a given tumor, towhich they may be directly administered, but also to prevent or treatcancer metastasis.

Several of the viruses of the invention are herpes simplex viruses (HSV)that include an inactivating mutation in the ICP47 locus of the virus.This mutation can occur, for example, between the BstEII site and theEcoNI site of the BamHI x fragment of HSV-1, and may comprise, e.g.,deletion of the BstEII-ExoNI fragment. Optionally, a herpes simplexvirus including a mutation between the BstEII and EcoNI sites can alsoinclude additional mutations. For example, such a virus can include aninactivating mutation in the γ34.5 neurovirulence determination locus ofthe virus, and/or an inactivating mutation elsewhere in the genome,e.g., in the ICP6 locus. The invention also includes herpes simplexviruses that include inactivating mutations in the ICP47 locus, in theabsence of an inactivating mutation in the γ34.5 neurovirulence locus.Optionally, such a virus can include an inactivating mutation inanother, non-γ34.5 neurovirulence locus, e.g., in the ICP6 locus.

The invention includes additional viruses that are based on herpesviruses, such as herpes simplex (HSV viruses), for example, HSV-1 (e.g.,HSV-1 strain F or strain Patton) or HSV-2, that include an inactivatingmutation in a virulence gene. In the case of herpes simplex viruses,this mutation can be an inactivating mutation in the γ34.5 gene, whichis the major HSV neurovirulence determinant. (See, e.g., FIG. 1 fordetails concerning the construction of examples of viruses that areincluded in the invention.)

In addition to the γ34.5 mutation, in one example, the viruses of theinvention can include a modification that results in early expression ofUS11, in the absence of an ICP-47-inactivating mutation in the BamHI xfragment of the vector. US11 is normally expressed as a true-late gene,requiring DNA replication for its expression. However, early expressionof US11 in some of the viruses of the invention can compensate for theγ34.5 defect by preventing the PKR-mediated shut-off of proteinsynthesis (see, e.g., FIG. 1E). Early expression of US11 in such a viruscan be achieved by, for example, inserting an early-acting promoterupstream of the US11 gene (FIG. 1E). Such promoters can include, forexample, the human cytomegalovirus (CMV) IE promoter, an HSV-1 IEpromoter, an HSV-1 E promoter, or any other heterologous promoter thatis active before the onset of DNA replication in the HSV-1 genome (see,e.g., below). An alternative approach to achieving early expression ofUS11 included in the invention involves inserting an exogenous copy of aUS11 gene elsewhere in the viral genome, under the control of anysuitable promoter that is active early in infection, such as one ofthose listed above, for example.

An additional HSV-based virus included in the invention includes, inaddition to an inactivating mutation in the γ34.5 locus, a secondmodification that results in downregulation of ICP47 expression, in theabsence of a mutation in the BamHI x fragment of the virus. In oneexample of such a virus, ICP47 coding sequences are fused with sequencesthat encode a peptide that prevents functional expression of ICP47 (see,e.g., FIG. 1E). Such a peptide can include, for example, a PESTsequence, which is rich in proline (P), glutamate (E), serine (S), andthreonine (T), and thus provides intramolecular signals for rapidproteolytic degradation (Rechsteiner et al., Trends Biochem. Sci.21(7):267-271, 1996). Such a poison sequence can be inserted into thevirus at, for example, the BstEII site, upstream of a strong promoterdriving US11 (FIG. 1E). In an alternative vector, signals that directRNA degradation are incorporated into the virus, to direct degradationof ICP47 RNA.

Other viruses included in the invention can include, in addition to aninactivating mutation in the γ34.5 locus, two additional modifications.The first additional modification results in early expression of US11and the second modification results in downregulation of ICP47expression, as described above, in the absence of a mutation in theBamHI x fragment of the virus. In one example of such a virus, anearly-expressing promoter is inserted upstream of the US11 gene andICP47 coding sequences are fused with sequences encoding a poisonsequence, such as a PEST sequence (FIG. 1E).

Any of the viruses described above and herein and elsewhere can includean additional mutation or modification that is made to prevent reversionof the virus to wild type. For example, the virus can include a mutationin the ICP6 gene (see below), which encodes the large subunit ofribonucleotide reductase. A specific example of a virus that is includedin the invention, G47Δ, is described in further detail below. Briefly,this virus includes a deletion in the γ34.5 gene, an inactivatinginsertion in the ICP6 gene, and a 312 basepair deletion in the ICP47gene.

The viruses described herein can be generated from any herpes virusfamily member, such as a neurotrophic, B-lymphotrophic, orT-lymphotrophic herpes virus. For example, a herpes simplex virus (HSV),such as HSV-1 or HSV-2, can be used. Alternatively, any of the followingviruses can be used: Varicella-zoster virus (VZV), herpes virus 6(HSV-6), Epstein Barr virus, cytomegalovirus, HHV6, and HHV7. Themethods and viruses described herein are described primarily inreference to HSV-1, but these methods can readily be applied to any ofthese other viruses by one of skill in this art.

As is noted above, the viruses of the invention can be used to treatcancer, as these viruses replicate in, and thus destroy dividing cells,such as cancer cells, but are avirulent to other cells. Examples ofcancer cells that can be destroyed, according to the invention, includecancer cells of nervous-system type tumors, for example, astrocytoma,oligodendroglioma, meningioma, neurofibroma, glioblastoma, ependymoma,Schwannoma, neurofibrosarcoma, neuroblastoma, pituitary tumor (e.g.,pituitary adenoma), and medulloblastoma cells. Other types of tumorcells that can be killed, pursuant to the present invention, include,for example, melanoma, prostate carcinoma, renal cell carcinoma,pancreatic cancer, breast cancer, lung cancer, colon cancer, gastriccancer, fibrosarcoma, squamous cell carcinoma, neurectodermal, thyroidtumor, lymphoma, hepatoma, mesothelioma, and epidermoid carcinoma cells,as well as other cancer cells mentioned herein. Also as is noted above,the viruses of the invention, which induce a systemic immune response tocancer, can be used to prevent or to treat cancer metastasis.

Other therapeutic applications in which killing of a target cell isdesirable include, for example, ablation of keratinocytes and epithelialcells responsible for warts, ablation of cells in hyperactive organs(e.g., thyroid), ablation of fat cells in obese patients, ablation ofbenign tumors (e.g., benign tumors of the thyroid or benign prostatichypertrophy), ablation of growth hormone-producing adenohypophysealcells to treat acromegaly, ablation of mammotropes to stop theproduction of prolactin, ablation of ACTH-producing cells to treatCushing's disease, ablation of epinephrine-producing chromaffin cells ofthe adrenal medulla to treat pheochromocytoma, and ablation ofinsulin-producing beta islet cells to treat insulinoma. The viruses ofthe invention can be used in these applications as well.

The effects of the viruses of the invention can be augmented if theviruses also contain a heterologous nucleic acid sequence encoding oneor more therapeutic products, for example, a cytotoxin, animmunomodulatory protein (i.e., a protein that either enhances orsuppresses a host immune response to an antigen), a tumor antigen, anantisense RNA molecule, or a ribozyme. Examples of immunomodulatoryproteins include, e.g., cytokines (e.g., interleukins, for example, anyof interleukins 1-15, α, β, or γ-interferons, tumor necrosis factor,granulocyte macrophage colony stimulating factor (GM-CSF), macrophagecolony stimulating factor (M-CSF), and granulocyte colony stimulatingfactor (G-CSF)), chemokines (e.g., neutrophil activating protein (NAP),macrophage chemoattractant and activating factor (MCAF), RANTES, andmacrophage inflammatory peptides MIP-1a and MIP-1b), complementcomponents and their receptors, immune system accessory molecules (e.g.,B7.1 and B7.2), adhesion molecules (e.g., ICAM-1, 2, and 3), andadhesion receptor molecules. Examples of tumor antigens that can beproduced using the present methods include, e.g., the E6 and E7 antigensof human papillomavirus, EBV-derived proteins (Van der Bruggen et al.,Science 254:1643-1647, 1991), mucins (Livingston et al., Curr. Opin.Immun. 4(5):624-629, 1992), such as MUC1 (Burchell et al., Int. J.Cancer 44:691-696, 1989), melanoma tyrosinase, and MZ2-E (Van derBruggen et al., supra). (Also see WO 94/16716 for a further descriptionof modification of viruses to include genes encoding tumor antigens orcytokines.)

As is noted above, the therapeutic product can also be an RNA molecule,such as an antisense RNA molecule that, by hybridization interactions,can be used to block expression of a cellular or pathogen mRNA.Alternatively, the RNA molecule can be a ribozyme (e.g., a hammerhead ora hairpin-based ribozyme) designed either to repair a defective cellularRNA, or to destroy an undesired cellular or pathogen-encoded RNA (see,e.g., Sullenger, Chem. Biol. 2(5):249-253, 1995; Czubayko et al., GeneTher. 4(9):943-949, 1997; Rossi, Ciba Found. Symp. 209:195-204, 1997;James et al., Blood 91(2):371-382, 1998; Sullenger, Cytokines Mol. Ther.2(3):201-205, 1996; Hampel, Prog. Nucleic Acid Res. Mol. Bio. 58:1-39,1998; Curcio et al., Pharmacol. Ther. 74(3):317-332, 1997).

A heterologous nucleic acid sequence can be inserted into a virus of theinvention in a location that renders it under the control of aregulatory sequence of the virus. Alternatively, the heterologousnucleic acid sequence can be inserted as part of an expression cassettethat includes regulatory elements, such as promoters or enhancers.Appropriate regulatory elements can be selected by those of ordinaryskill in the art based on, for example, the desired tissue-specificityand level of expression. For example, a cell-type specific ortumor-specific promoter can be used to limit expression of a geneproduct to a specific cell type. This is particularly useful, forexample, when a cytotoxic, immunomodulatory, or tumor antigenic geneproduct is being produced in a tumor cell in order to facilitate itsdestruction. In addition to using tissue-specific promoters, localadministration of the viruses of the invention can result in localizedexpression and effect.

Examples of non-tissue specific promoters that can be used in theinvention include the early Cytomegalovirus (CMV) promoter (U.S. Pat.No. 4,168,062) and the Rous Sarcoma Virus promoter (Norton et al.,Molec. Cell. Biol. 5:281, 1985). Also, HSV promoters, such as HSV-1 IEand IE 4/5 promoters, can be used.

Examples of tissue-specific promoters that can be used in the inventioninclude, for example, the prostate-specific antigen (PSA) promoter,which is specific for cells of the prostate; the desmin promoter, whichis specific for muscle cells (Li et al., Gene 78:243, 1989; Li et al.,J. Biol. Chem. 266:6562, 1991; Li et al., J. Biol. Chem. 268:10403,1993); the enolase promoter, which is specific for neurons (Forss-Petteret al., J. Neuroscience Res. 16(1):141-156, 1986); the β-globinpromoter, which is specific for erythroid cells (Townes et al., EMBO J.4:1715, 1985); the tau-globin promoter, which is also specific forerythroid cells (Brinster et al., Nature 283:499, 1980); the growthhormone promoter, which is specific for pituitary cells (Behringer etal., Genes Dev. 2:453, 1988); the insulin promoter, which is specificfor pancreatic β cells (Selden et al., Nature 321:545, 1986); the glialfibrillary acidic protein promoter, which is specific for astrocytes(Brenner et al., J. Neurosci. 14:1030, 1994); the tyrosine hydroxylasepromoter, which is specific for catecholaminergic neurons (Kim et al.,J. Biol. Chem. 268:15689, 1993); the amyloid precursor protein promoter,which is specific for neurons (Salbaum et al., EMBO J. 7:2807, 1988);the dopamine β-hydroxylase promoter, which is specific for noradrenergicand adrenergic neurons (Hoyle et al., J. Neurosci. 14:2455, 1994); thetryptophan hydroxylase promoter, which is specific for serotonin/pinealgland cells (Boularand et al., J. Biol. Chem. 270:3757, 1995); thecholine acetyltransferase promoter, which is specific for cholinergicneurons (Hersh et al., J. Neurochem. 61:306, 1993); the aromatic L-aminoacid decarboxylase (AADC) promoter, which is specific forcatecholaminergic/5-HT/D-type cells (That et al., Mol. Brain Res.17:227, 1993); the proenkephalin promoter, which is specific forneuronal/spermatogenic epididymal cells (Borsook et al., Mol.Endocrinol. 6:1502, 1992); the reg (pancreatic stone protein) promoter,which is specific for colon and rectal tumors, and pancreas and kidneycells (Watanabe et al., J. Biol. Chem. 265:7432, 1990); and theparathyroid hormone-related peptide (PTHrP) promoter, which is specificfor liver and cecum tumors, and neurilemoma, kidney, pancreas, andadrenal cells (Campos et al., Mol. Rnfovtinol. 6:1642, 1992).

Examples of promoters that function specifically in tumor cells includethe stromelysin 3 promoter, which is specific for breast cancer cells(Basset et al., Nature 348:699, 1990); the surfactant protein Apromoter, which is specific for non-small cell lung cancer cells (Smithet al., Hum. Gene Ther. 5:29-35, 1994); the secretory leukoproteaseinhibitor (SLPI) promoter, which is specific for SLPI-expressingcarcinomas (Garver et al., Gene Ther. 1:46-50, 1994); the tyrosinasepromoter, which is specific for melanoma cells (Vile et al., GeneTherapy 1:307, 1994; WO 94/16557; WO 93/GB1730); the stress induciblegrp78/BiP promoter, which is specific for fibrosarcoma/tumorigenic cells(Gazit et al., Cancer Res. 55(8):1660, 1995); the AP2 adipose enhancer,which is specific for adipocytes (Graves, J. Cell. Biochem. 49:219,1992); the α-1 antitrypsin transthyretin promoter, which is specific forhepatocytes (Grayson et al., Science 239:786, 1988); the interleukin-10promoter, which is specific for glioblastoma multiform cells (Nitta etal., Brain Res. 649:122, 1994); the c-erbB-2 promoter, which is specificfor pancreatic, breast, gastric, ovarian, and non-small cell lung cells(Harris et al., Gene Ther. 1:170, 1994); the α-B-crystallin/heat shockprotein 27 promoter, which is specific for brain tumor cells (Aoyama etal., Int. J. Cancer 55:760, 1993); the basic fibroblast growth factorpromoter, which is specific for glioma and meningioma cells (Shibata etal., Growth Fact. 4:277, 1991); the epidermal growth factor receptorpromoter, which is specific for squamous cell carcinoma, glioma, andbreast tumor cells (Ishii et al., Proc. Natl. Acad. Sci. U.S.A. 90:282,1993); the mucin-like glycoprotein (DF3, MUC1) promoter, which isspecific for breast carcinoma cells (Abe et al., Proc. Natl. Acad. Sci.U.S.A. 90:282, 1993); the mts1 promoter, which is specific formetastatic tumors (Tulchinsky et al., Proc. Natl. Acad. Sci. U.S.A.89:9146, 1992); the NSE promoter, which is specific for small-cell lungcancer cells (Forss-Petter et al., Neuron 5:187, 1990); the somatostatinreceptor promoter, which is specific for small cell lung cancer cells(Bombardieri et al., Eur. J. Cancer 31A:184, 1995; Koh et al., Int. J.Cancer 60:843, 1995); the c-erbB-3 and c-erbB-2 promoters, which arespecific for breast cancer cells (Quin et al., Histopathology 25:247,1994); the c-erbB4 promoter, which is specific for breast and gastriccancer cells (Rajkumar et al., Breast Cancer Res. Trends 29:3, 1994);the thyroglobulin promoter, which is specific for thyroid carcinomacells (Mariotti et al., J. Clin. Endocrinol. Meth. 80:468, 1995); theα-fetoprotein promoter, which is specific for hepatoma cells (Zuibel etal., J. Cell. Phys. 162:36, 1995); the villin promoter, which isspecific for gastric cancer cells (Osborn et al., Virchows Arch. A.Pathol. Anat. Histopathol. 413:303, 1988); and the albumin promoter,which is specific for hepatoma cells (Huber, Proc. Natl. Acad. Sci.U.S.A. 88:8099, 1991).

As is noted above, the viruses of the invention can be used in in vivomethods, for example, to kill a cell and/or to introduce a therapeuticgene product into the cell. To carry out these methods, the viruses ofthe invention can be administered by any conventional route used inmedicine. For example, a virus of the invention can be administereddirectly into a tissue in which an effect, e.g., cell killing and/ortherapeutic gene expression, is desired, for example, by directinjection or by surgical methods (e.g., stereotactic injection into abrain tumor; Pellegrino et al., Methods in Psychobiology (AcademicPress, New York, N.Y., 67-90, 1971)). An additional method that can beused to administer vectors into the brain is the convection methoddescribed by Bobo et al. (Proc. Natl. Acad. Sci. U.S.A. 91:2076-2080,1994) and Morrison et al. (Am. J. Physiol. 266:292-305, 1994). In thecase of tumor treatment, as an alternative to direct tumor injection,surgery can be carried out to remove the tumor, and the vectors of theinvention inoculated into the resected tumor bed to ensure destructionof any remaining tumor cells. Alternatively, the vectors can beadministered via a parenteral route, e.g., by an intravenous,intraarterial, intracerebroventricular, subcutaneous, intraperitoneal,intradermal, intraepidermal, or intramuscular route, or via a mucosalsurface, e.g., an ocular, intranasal, pulmonary, oral, intestinal,rectal, vaginal, or urinary tract surface.

Any of a number of well-known formulations for introducing viruses intocells in mammals, such as humans, can be used in the invention. (See,e.g., Remington's Pharmaceutical Sciences (18^(th) edition), ed. A.Gennaro, 1990, Mack Publishing Co., Easton, Pa.) However, the virusescan be simply diluted in a physiologically acceptable solution, such assterile saline or sterile buffered saline, with or without an adjuvantor carrier.

The amount of virus to be administered depends, e.g., on the specificgoal to be achieved, the strength of any promoter used in the virus, thecondition of the mammal (e.g., human) intended for administration (e.g.,the weight, age, and general health of the mammal), the mode ofadministration, and the type of formulation. In general, atherapeutically or prophylactically effective dose of, e.g., from about10¹ to 10¹⁰ plaque forming units (pfu), for example, from about 5×10⁴ to1×10⁶ pfu, e.g., from about 1×10⁵ to about 4×10⁵ pfu, although the mosteffective ranges may vary from host to host, as can readily bedetermined by one of skill in this art. Also, the administration can beachieved in a single dose or repeated at intervals, as determined to beappropriate by those of skill in this art.

A specific example of a virus of the invention, designated G47Δ, whichis a new, multimutated, replication-competent HSV-1 virus, derived fromG207 by a deletion within the non-essential α47 gene (Mavromara-Nazos etal., J. Virol. 60:807-812, 1986), is now described. Because of theoverlapping transcripts encoding ICP47 and US11 (FIG. 2B), the deletionin α47 also places the late US11 gene under control of theimmediate-early α47 promoter. This enhances the growth properties ofγ34.5⁻ mutants by precluding the shutoff of protein synthesis (Mohr etal., EMBO J. 15:4759-4766, 1996; He et al., J. Virol. 71:6049-6054,1997; Cassady et al., J. Virol. 72:7005-7011, 1998; Cassady et al., J.Virol. 72:8620-8626, 1998). Nevertheless, we found that G47Δ was as safeas G207, which is now in clinical trials in humans, when inoculated intothe brains of A/J mice at 2×10⁶ pfu. We show here that human melanomacells infected with G47Δ were more effective at stimulating theirmatched tumor-infiltrating lymphocytes (TILs) than those infected withG207, that G47Δ showed enhanced replication in cultured tumor cells, andthat G47Δ was more efficacious than G207 at inhibiting tumor growth inboth human xenograft and mouse syngeneic tumor models tested. Ourresults show that G47Δ can be used for tumor therapy. Additional detailsof this virus and its properties are provided as follows.

Experimental Results

Construction and Replication of G47Δ

G47Δ was constructed by deleting 312 basepairs from G207 in the U_(S)region adjacent to TR_(S) (FIG. 2). Southern blot analyses of G47Δ DNAconfirmed the presence of a 0.3 kilobase deletion in the α47 gene and a1 kilobase deletion in the γ34.5 gene. R47Δ, with the same deletion inthe α47 locus, was generated from R3616, the parental virus of G207 thathas an active ribonucleotide reductase (Chou et al., Science250:1262-1266, 1990).

To investigate the effects of the α47 deletion on the growth propertiesof γ34.5-deficient mutants (G207 and R3616), we determined the yield ofprogeny virus following infection of human tumor cells lines SK-N-SH(neuroblastoma), U87MG (glioma), U373MG (glioma), and SQ20B (head andneck squamous cell carcinoma). By 24 hours post-infection at a low MOI,G47Δ produced higher yields than G207, resulting in an approximately 4to 1000-fold increase in titer (FIG. 3). In a single-step growthexperiment in U87MG cells (MOI=2), the virus yield of G47Δ was 12 timesgreater than with G207. R47Δ similarly yielded higher titers than itsparent R3616 in all tumor cell lines tested; however, neither G47Δ norR47Δ grew as well as wild-type parental strain F. To determine whethervirus yields were affected by cell density, Vero and SK-H-SH cells wereseeded at normal or high density (8×10⁵ or 1.6×10⁶ cells/well), infectedwith strain F, G207, or G47Δ at a MOI of 0.01, and harvested 48 hourspost-infection. G47Δ produced a higher yield in the high-densityculture, as opposed to G207, which had a reduced yield. The ability togenerate higher yields of G47Δ in Vero cells facilitates manufacturingof high titer stocks for clinical use.

Cytopathic Effect of G47Δ In Vitro

The cytolytic activity of G47Δ in vitro was compared to that of G207 invarious neural crest-derived tumor cell lines. In human cell lines,U87MG and melanomas 624 and 888, G47Δ killed tumor cells significantlymore rapidly than G207 at a low MOI of 0.01 (FIG. 4). At a MOI of 0.1,both G207 and G47Δ killed all the cells within 1-3 days of infection.Neuro2a, a murine neuroblastoma cell line, was resistant to killing byboth G207 and G47Δ at a MOI of 0.01. At a MOI of 0.1, G47Δ wassignificantly more efficient at destroying tumor cells than G207 (FIG.4), an effect also seen with N18 mouse neuroblastoma cells. We havefound that mouse tumor cells are generally more resistant to G207replication than human tumor cells (Todo et al., Hum. Gene Ther.10:2741-2755, 1999; Toda et al., Hum. Gene Ther. 10:385-393, 1999; Todoet al., Cancer Res. 61:153-161, 2001).

MHC Class I Expression in G47Δ-Infected Cells

ICP47 inhibits the function of TAP in translocating peptides across theendoplasmic reticulum in human cells, but not in mouse or rat cells (Ahnet al., EMBO J. 15:3247-3255, 1996; Tomazin et al., J. Virol.72:2560-2563, 1998). Because G47Δ lacks ICP47, infected cells shouldhave levels of MHC class I expression typical of uninfected cells. Weexamined MHC class I down-regulation in Detroit 551 human diploidfibroblasts using flow cytometric analyses for human lymphocyte antigenclass I (HLA-1). At 48 hours post-infection, all cells infected withHSV-1 containing an intact α47 gene (strain F, G207, and R3616) showed adecrease in cell surface MHC class I, resulting in approximately 40% inpeak levels compared to mock-infected control cells (FIGS. 5A and 5B).By contrast, there was no down-regulation in G47Δ infected cells (FIG.5A). In R47Δ-infected cells, MHC class I expression remained higher thanin strain F or R3616-infected cells, but was reduced compared to G47Δ(˜75% of mock-infected peak levels). Studies at different time points(6, 24, and 48 hours post-infection) revealed that differences in MHCclass I down-regulation between ICP47 expressing (G207 and R3616) andnon-expressing (G47Δ and R47Δ) infected cells did not become apparentuntil after 6 hours post-infection (FIG. 5B).

Infection of human melanoma cells with G47Δ also resulted in higherlevels of MHC class I expression than with G207, although the preclusionof down-regulation was partial. In general, a greater effect wasobserved in cell lines with high basal levels of MHC class I (938 and1102) compared to those with low levels of MHC class I (624, 888, and1383) (FIG. 5C).

G47Δ-Infected Human Melanoma Cells Stimulate Human T Cells In Vitro

Three human melanoma cell lines were tested for their abilities tostimulate the matched TIL lines after G47Δ infection (888 and 1102 withTIL888 (Robbins et al., Cancer Res. 54:3124-3126, 1994)), and 938 withTIL1413 (Kang et al., J. Immunol. 155:1343-1348, 1995). G47Δ-infected1102 melanoma cells, with the highest level of MHC class I expression,caused a better stimulation of TIL cells compared to G207-infectedcells, resulting in 41% more IFN-γ secretion (FIG. 6). There wasessentially no stimulation of this same TIL line with G47Δ orG207-infected 888 melanoma cells, which had very low levels of MHC classI expression. G47Δ-infected 938 melanoma cells stimulated TIL1413 cells,causing an increase in IFN-γ secretion that was not statisticallysignificant. The results demonstrate that the higher MHC class Iexpression that may ensue in G47Δ versus G207-infected cells can enhanceT cell stimulation.

Antitumor Efficacy of G47Δ In Vivo

In a human xenograft model, athymic mice harboring establishedsubcutaneous U87MG glioma tumors (approximately 6 mm in diameter),intraneoplastic inoculation of G207 or G47Δ (10⁶ pfu) followed by asecond inoculation 3 days later caused a significant reduction in U87MGtumor growth (p<0.05 and p<0.001 versus control on day 24, respectively;unpaired t test; FIG. 7). G47Δ treatment was significantly moreefficacious than G207, resulting in reduced average tumor volumes (FIG.7). This was reflected in the prolonged survival of animals and numberof ‘cures’ (complete tumor regression with no tumor regrowth during a3-month follow up) (Table 1). At the dose tested, survival wassignificantly prolonged in the G207-treatment group (p<0.05 versus mock,Wilcoxon test), and to an even greater extent in the G47Δ-treatedanimals (p<0.05 versus G207, Wilcoxon test).

TABLE 1 Subcutaneous tumor therapy by G47Δ Number cured/total treatedTumor (Mouse) Mock G207 G47Δ U87MG (Athymic) 0/13 3/12 8/12*^(†) Neuro2a(A/J) 0/10 1/10 3/10 *p < 0.05 versus G207, ^(†)p < 0.001 versus Mock,Fisher's test.

The efficacy of G47Δ was further tested in an immunocompetent mousetumor model, subcutaneous, poorly immunogenic Neuro2a neuroblastomatumors in syngeneic A/J mice. Established tumors of approximately 6 mmin diameter were inoculated with mock, G207, or G47Δ (10⁶ pfu) on days 0and 3. Again, while both G207 and G47Δ caused a significant reduction inNeuro2a tumor growth (p<0.05 and p<0.001 versus control on day 15,respectively; unpaired t test), the efficacy of G47Δ was greater thanthat of G207 (FIG. 7). Kaplan-Meier analysis demonstrated that G207 atthis dose did not significantly extend the survival of Neuro2atumor-bearing A/J mice, whereas G47Δ significantly prolonged survival ofthe animals compared with mock and G207 (p<0.01 and p<0.05,respectively, Wilcoxon test). In a 3.5-month follow-up period, there wasan increased number of ‘cures’ among the G47Δ-treated mice (notstatistically significant, Fisher's test; Table 1).

Safety of G47Δ with Intracerebral Inoculation

To evaluate the toxicity of G47Δ in the brain, A/J mice were inoculatedintracerebrally with mock, strain F (2×10³ pfu), G207 (2×10⁶ pfu), orG47Δ (2×10⁶ pfu). This dose was the highest dose obtainable for G207 inthe volume injected. Each mouse was monitored daily for clinicalmanifestations for 3 weeks. All 8 mock-inoculated mice survived withoutany abnormal manifestations, whereas all 10 strain F-inoculated micedeteriorated rapidly and became moribund within 7 days of inoculation.All 8 G207-inoculated mice and 10 G47Δ-inoculated mice survived. Two ofthe G207-inoculated mice and 1 G47Δ-inoculated mouse temporarilymanifested (3-6 days post-inoculation) slight hunching or a slightlysluggish response to external stimuli. This shows that G47Δ is as safeas G207 when inoculated in the brain of A/J mice at this dose.

The results described above were obtained using the following Materialsand Methods.

Materials and Methods

Cells

Vero (African green monkey kidney), SK-N-SH (human neuroblastoma), U87MG(human glioma), U373MG (human glioma), Neuro2a (murine neuroblastoma),and Detroit 551 (diploid human fibroblast) cell lines were purchasedfrom American Type Culture Collection (Rockville, Md.). SQ20B (head andneck squamous cell carcinoma) cells were provided by Dr. R. Weichselbaum(University of Chicago, Chicago, Ill.). N18 murine neuroblastoma cellswere provided by Dr. K. Ikeda (Tokyo Institute of Psychiatry, Tokyo,Japan). Human melanoma cell lines 624, 888, 938, 1102, and 1383, andhuman T cell lines TIL888 and TIL1413, were provided by Dr. J.Wunderlich (NIH, Bethesda, Md.). All tumor cells were maintained inDulbecco's modified Eagle medium supplemented with 10% fetal calf serum(FCS), 2 mM glutamine, penicillin (100 U/ml), streptomycin (100 μg/ml),and 2.5 μg/ml Fungizone. Human T cells were maintained in AIM-V medium(Gibco BRL, Life Technologies, Rockville, Md.) supplemented with 10%human serum (type AB, Rh⁺; Valley Biomedical Products, Winchester, Va.),interleukin 2 (600 international units (IU)/ml, Chiron Corporation,Emeryville, Calif.), penicillin (50 U/ml), and 1.25 μg/ml Fungizone.

Generation of G47 Δ

Plasmid pIE12 contains an 1818 basepair BamHI-EcoRI fragment from theHSV-1 BamHI x fragment, which encompasses the ICP47 coding region(Johnson et al., J. Virol. 68:6347-6362, 1994). A 312 basepair fragmentcontaining the ICP47 coding region between the BstEII and EcoNI siteswas deleted from pIE12 to create pIE12 Δ (FIG. 2C). Vero cells wereseeded on 6-well dishes at a density of 1−2×10⁵ cells per well.Transfections were performed using a range of DNA concentrations from 1to 3 μg, including a 1:1:1 mixture of G207 DNA (Mineta et al., Nat. Med.1:938-943, 1995), pIE12 (intact), and pIE12 Δ cleaved with BamHI andXhoI, with 8 μl LipofectAMINE™ (Life Technologies), according to themanufacturer's instructions. The viral progeny from the transfectionwere then passaged twice in SK-N-SH cells to enrich for recombinantsthat contained a deletion in ICP47 as follows. SK-N-SH cells were seededat a density of 5×10⁶ cells per 10 cm dish, infected the following dayat a range of MOI's from 0.01 to 1 pfu per cell, and harvested at 48hours post-infection. This process was then repeated. The deletion inpIE12 Δ was designed to generate a second-site suppressor mutation ofγ34.5 in the virus, and thus permit growth of successful recombinants onSK-N-SH cells (Mohr et al., EMBO J. 15:4759-4766, 1996). Individualplaques from SK-N-SH-enriched stocks were plaque-purified on Vero cellsunder agarose overlays and screened for the presence of the deletion inICP47 by Southern blotting. A stock was prepared from one individualplaque that was homogeneous for the ICP47 deletion and designated asG47Δ. R47Δ was constructed similarly, except R3616 (Chou et al., Science250:1262-1266, 1990) DNA was used in place of G207 DNA (R3616 wasprovided by Dr. B. Roizman, University of Chicago, Chicago, Ill.). Virustitration was performed as previously described (Miyatake et al., J.Virol. 71:5124-5132, 1997).

Virus Yield Studies

Cells were seeded on 6-well plates at 5×10⁵, 8×10⁵, or 1.6×10⁶ cells perwell. Triplicate or duplicate wells were infected with the viruses 6-8hours after seeding at a MOI of 0.01. At 24 or 48 hours post-infection,the cells were scraped into the medium and lysed by three cycles offreeze-thawing. The progeny virus was titered as previously describedwith a modification (Miyatake et al., J. Virol. 71:5124-5132, 1997).Briefly, Vero cells were plated in 6-well plates at 8×10⁵ cells/well.After 4-8 hours incubation at 37° C., cells were infected in 1 ml growthmedium at 37° C. overnight, after which 1 ml medium containing 0.4%human IgG (ICN Pharmaceuticals) was added. Wells were incubated at 37°C. for another 2 days, and the number of plaques was counted afterstaining with methylene blue (0.5% w/v in 70% methanol).

In Vitro Cytotoxicity Studies

In vitro cytotoxicity studies were performed as previously described(Todo et al., Hum. Gene Ther. 10:2741-2755, 1999), with a modificationfor human melanoma cells, which were grown in medium containing 10% FCS.The number of surviving cells was counted daily with a Coulter counter(Beckman Coulter, Fullerton, Calif.) and expressed as a percentage ofmock-infected controls.

Flow Cytometric Analyses

Cells were plated in 6 well plates at 1×10⁶ cells/well and infected withvirus (MOI=3) 24 hours after seeding. Cells were incubated in thepresence of ganciclovir (200 ng/ml) at 39.5° C. for 6, 24, or 48 hours,harvested by trypsinization, and washed once with 2 ml PBS. G207 andG47Δ contain temperature-sensitive mutations in ICP4, so they canreplicate at 37° C., but not at 39.5° C. (Mineta et al., Nat. Med.1:938-943, 1995. Approximately 5×10⁵ cells were then used for flowcytometric analyses using FITC-conjugated anti-human HLA class I antigen(clone W6/32, Sigma, St. Louis, Mo.) and performed as previouslydescribed.

Human T Cell Stimulation Assays

Human melanoma cells (888, 938, or 1102) were plated in 6 well plates at5×10⁵ cells/well, and infected with G207 or G47Δ (MOI=3), or withoutvirus (mock) 24 hours after seeding. Cells were incubated in growthmedium containing 10% FCS and ganciclovir (200 ng/ml) at 39.5° C. for 3hours (888) or 6 hours (938 and 1102). Cells were then harvested byscraping, and a portion was used for cell counting. Infected melanomacells (1×10⁵) were then co-cultured with an equal number of respondinghuman T cells in 200 μl AIM-V medium containing ganciclovir (200 ng/ml)in a flat-bottom 96-well plate. Melanomas 888 and 1102 were co-culturedwith TIL888 cells, and melanoma 938 was cultured with TIL1413 cells. TILlines 888 and 1413 both recognize tyrosinase, a melanoma antigen, in anHLA-A24 restricted fashion (Robbins et al., Cancer Res. 54:3124-3126,1994; Kang et al., J. Immunol. 155:1343-1348, 1995). After an 18 hourincubation at 37° C., the plate was centrifuged at 800 g for 10 minutes,and conditioned medium was collected. IFN-γ concentrations were measuredby enzyme-linked immunosorbent assay using a human IFN-γ ELISA kit(Endogen, Woburn, Mass.). The IFN-γ measurements in TIL cells withoutstimulator cells were considered the base release levels and used tocalculate the increase of IFN-γ secretion in stimulated TIL cells.

Animal Studies

Six-week-old female A/J mice and athymic nude mice (BALB/c nu/nu) werepurchased from the National Cancer Institute (Frederick, Md.), and cagedin groups of four or less. Subcutaneous tumor therapy was performed aspreviously described (Todo et al., Hum. Gene Ther. 10:2741-2755, 1999;Todo et al., Cancer Res. 61:153-161, 2001).

Intracerebral Inoculation Toxicity Studies

Mock (PBS containing 10% glycerol), strain F (2×10³ pfu), G207 (2×10⁶pfu), or G47Δ (2×10⁶ pfu) in a volume of 5 μl was injected over 5minutes into the right hemisphere of the brains of 6-week-old female A/Jmice (n=8, 10, 8, and 10, respectively) using a KOPF stereotactic frame.Cages were then blinded and mice monitored daily for clinicalmanifestations for 3 weeks.

All references cited herein are incorporated by reference in theirentirety. Other embodiments are within the following claims.

What is claimed is:
 1. A method of treating melanoma in a patient, saidmethod comprising administering to a patient having melanoma a herpessimplex virus-1 comprising an inactivating mutation in the ICP47 locusof said virus that results in early expression of US11, and aninactivating mutation in the γ34.5 neurovirulence locus of said virus.2. The method of claim 1, wherein the patient has or is at risk ofdeveloping one or more metastases.
 3. The method of claim 1, whereinsaid herpes simplex virus is administered to a tumor of said patient. 4.The method of claim 1, wherein said herpes simplex virus is administeredparenterally to said patient.
 5. The method of claim 1, wherein saidearly expression of US11 is a result of the US11 gene being placed underthe control of an early-expressing promoter.
 6. The method of claim 5,wherein said early-expressing promoter is the ICP 47 promoter of saidvirus.
 7. The method of claim 1, wherein said inactivating mutation inthe ICP47 locus of said virus comprises a mutation within theBstEII-EcoNI fragment of the BamHI x fragment of said virus.
 8. Themethod of claim 1, wherein said herpes simplex virus comprises aninactivating mutation in the ICP6 locus of said virus.
 9. The method ofclaim 1, wherein said herpes simplex virus further comprises sequencesencoding a heterologous gene product.
 10. The method of claim 9, whereinsaid heterologous gene product comprises a vaccine antigen or animmunomodulatory protein.
 11. The method of claim 10, wherein saidimmunomodulatory protein is selected from the group consisting of acytokine, a chemokine, RANTES, a macrophage inflammatory peptide, acomplement component or receptor, an immune system accessory molecules,an adhesion molecule, and an adhesion receptor molecule.
 12. The methodof claim 11, wherein said cytokine is selected from the group consistingof an interleukin, tumor necrosis factor, granulocyte macrophage colonystimulating factor (GM-CSF), macrophage colony stimulating factor(M-CSF), and granulocyte colony stimulating factor (G-CSF).